Production Of Transgenic Fish Research Articles

Insights into the control and consequences of breathing adjustments in fishes-from larvae to adults.

Adjustments of ventilation in fishes to regulate the volume of water flowing over the gills are critically important responses to match branchial gas transfer with metabolic needs and to defend homeostasis during environmental fluctuations in O2 and/or CO2 levels. In this focused review, we discuss the control and consequences of ventilatory adjustments in fish, briefly summarizing ventilatory responses to hypoxia and hypercapnia before describing the current state of knowledge of the chemoreceptor cells and molecular mechanisms involved in sensing O2 and CO2. We emphasize, where possible, insights gained from studies on early developmental stages. In particular, zebrafish (Danio rerio) larvae have emerged as an important model for investigating the molecular mechanisms of O2 and CO2 chemosensing as well as the central integration of chemosensory information. Their value stems, in part, from their amenability to genetic manipulation, which enables the creation of loss-of-function mutants, optogenetic manipulation, and the production of transgenic fish with specific genes linked to fluorescent reporters or biosensors.

Влияние процесса микроинъекции на выживаемость эмбрионов зебрафиш (Danio rerio) при получении трансгенных рыб

Влияние процесса микроинъекции на выживаемость эмбрионов зебрафиш (Danio rerio) при получении трансгенных рыб

Zebrafish embryos as a model to study bacterial virulence.

In recent years the zebrafish has gained enormous attention in infection biology, and many protocols have been developed to study interaction of both human and fish pathogens, including viruses, fungi, and bacteria, with the host. Especially the extraordinary possibilities for live imaging of disease processes in the transparent embryos using fluorescent bacteria and cell-specific reporter fish combined with gene knockdown, transcriptome, and genetic studies have dramatically advanced our understanding of disease mechanisms. The zebrafish embryo is amenable to study virulence of both extracellular and facultative intracellular pathogens introduced through the technique of microinjection. Several protocols have been published that address the different sites of injection, antisense strategies, imaging, and production of transgenic fish in detail. Here we describe a protocol to study the virulence profiles, ranging from acute fatal to persistent, of bacteria belonging to the Burkholderia cepacia complex. This standard operating protocol combines simple survival assays, analysis of bacterial kinetics, analysis of the early innate immune response with qRT-PCR, and the use of transgenic reporter fish to study interactions with host phagocytes, and is also applicable to other pathogens.

The history of the aquaculture genetics program at Auburn University

The Auburn University Aquaculture Genetics Program has an 80-year history with focused efforts on genetics and breeding, transgenic technology and genomics being initiated 43, 27 and 18 years ago. This resulted in the first successful selection experiments for channel catfish, Ictalurus punctatus , the basis of the monosex tilapia industry, the first production of transgenic fish in the US, major advances in catfish genomics and the first production of a xenogenic catfish. The most significant impact has been the establishment of the technology to allow a hybrid catfish industry, which produced 150 million hybrid catfish fry in 2012. Paper presented at the International Symposium on Genetics in Aquaculture XI, Auburn, AL, June 24–30, 2012. ► History of Aquaculture Genetics. ► The establishment of the Aquaculture genetics program at Auburn University. ► Contributions made by scientists and Graduate students at Auburn University.

Endogenous Transposases Affect Differently Sleeping Beauty and Frog Prince Transposons in Fish Cells

Fish cells stably expressing exogenous genes have potential applications in the production of fish recombinant proteins, gene-function studies, gene-trapping, and the production of transgenic fish. However, expression of a gene of interest after random integration may be difficult to predict or control. In the past decade, major contributions have been made in vertebrate-gene transfer, by using tools derived from DNA transposons. Among them, the Sleeping Beauty (SB) and Frog Prince (FP) transposons, derived, respectively, from fish and frog genomes, mediate transposition in a large variety of cells, although with different efficiency. This study was aimed at assessing the activities of the SB and the FP transposases in fish cell lines from genetically distant species (CHSE-214, RTG-2, BF-2, EPC, and SAF-1). Their transpositional ability was evaluated by the plasmid-based excision assay, the colony formation assay, and the footprint patterns. The results reveal that while both transposases are active in all cell lines, the transposition rates and the precision of the transposition are overall higher with FP than SB. Our results also indicated a key role of cell-specific host factors in transposition, which was associated with the presence of Tc1-like endogenous transposases; this effect was more accentuated in the two salmonid cell lines transfected with SB. This result agrees with previous studies supporting the use of transposons in heterologous organisms to prevent from genomic instability and from impeding the precise activity of the exogenous transposase.

Transposon-mediated BAC transgenesis in zebrafish and mice

BackgroundBacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications.ResultsWe took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations.ConclusionThe Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.

Enhanced resistance to Aeromonas hydrophila infection and enhanced phagocytic activities in human lactoferrin-transgenic grass carp ( Ctenopharyngodon idellus)

Human lactoferrin (hLF) is an iron-binding protein with antimicrobial and immunomodulatory activities. hLF cDNA was transferred into grass carp via electroporated sperm. The production of transgenic fish was as high as 55% under the best parameters, 2 11 pulses and 20-min incubation. The expression of the transgene was demonstrated by the detection of hLF mRNA by RT-PCR. We also investigated the response of G 0 transgenic grass carp to Aeromonas hydrophila infection. Serum lysozyme activities ( P>0.05) and phagocytic activities of kidney cells ( P<0.05) were measured in transgenic individuals. The transgenic fish not only cleared A. hydrophila significantly faster than the control carp ( P<0.05), but also showed enhanced phagocytic activities. The result shows that hLF has immunomodulatory activities in hLF-transgenic grass carp. The transgenic grass carp exhibited enhanced immunity to A. hydrophila infection. These results reveal that the mechanisms of disease resistance are different between hLF-transgenic plants and hLF-transgenic grass carp.

High efficiency production of germ-line transgenic Japanese medaka (Oryzias latipes) by electroporation with direct current-shifted radio frequency pulses.

Although there have been several studies showing the production of transgenic fish through electroporation techniques, success rates have been low and few studies show germ-line integration and expression. When electroporation has been successful, the device used is no longer commercially available. The goal of this experiment was to find an alternative efficient method of generating transgenic Japanese medaka (Oryzias latipes) using a commercially available electroporation device. The Gene Pulser II and RF module (Bio-Rad Laboratories, USA), along with two reporter gene constructs, were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology, the Gene Pulser II incorporates a direct current (DC)-shifted radio frequency (RF) signal. With this technique, over 1000 embryos can be electroporated in less than 30 min. The plasmid pCMV-SPORT-beta-gal (Invitrogen, USA) was used in the supercoiled form to optimize parameters for gene transfer into single-celled embryos, and resulted in up to 100% somatic gene transfer. Similar conditions were used to generate fish transgenic for both the pCMV-EGFP plasmid (Clontech, USA) and a cytomegalovirus (CMV) driven phytase-EGFP construct. The conditions used were a voltage of 25 V, a percent modulation of 100%, a radio frequency of 35 kHz, a burst duration of 10 ms, 3 bursts, and a burst interval of 1.0 s. Seventy percent of the embryos electroporated with the pCMV-EGFP construct survived to sexual maturity, and of those, 85% were capable of passing the transgene on to their offspring. Transgenic second generation back-crossed (BC2) fry were subjected to Southern blot analysis, which confirmed germ-line integration, and observation for green fluorescence protein, which confirmed protein expression. DC-shifted RF pulses are effective and efficient in the production of transgenic medaka, and germ-line integration and expression can be achieved without linearization of the transgene vector.

Regulation and expression of transgenes in fish -- a review.

Transgenic fish, owing to a number of advantages which they offer over other species, are proving to be valuable model systems for the study of gene regulation and development genetics in addition to being useful targets for the genetic manipulation of commercially important traits. Despite having begun only a decade ago, the production of transgenic fish has become commonplace in a number of laboratories world-wide and considerable progress has been made. In this review, we initially consider the various regulatory elements and coding genes which have been used in fish, and subsequently discuss and compare both the transient and long-term fate and expression patterns of injected DNA sequences in the context of the different factors which are likely to have an effect on the expression of transgenes.

Gene transfer in fish: Potential and practice

Abstract During the past decade, developments in genetic engineering technology have led to the production of transgenic fish. In this paper, the impact of this novel strategy for genetic improvement of fish is examined in the context of possible benefits, probable limitations, and actual results. The role of gene transfer in improving fish growth rates, combatting disease, and altering other specific aspects of fish production is reviewed. Practical considerations, particularly in relation to the selection of a gene construct and its subsequent transfer, are outlined. A detailed discussion of the fate of transferred genes and the efficiency of transgenic fish production is also included. Finally, we review some of the practical precautions which must be observed if this novel technology is to safely complement conventional fish production strategies.

Implications of Introduction of Transgenic Fish into Natural Ecosystems

Production of transgenic fish has increased in scope, aimed at improving performance traits in economically important species or at creating model systems for basic biological problems. A variety of transgenic phenotypes will become possible as more genes are cloned and characterized. Categories of altered phenotypes include modified: metabolic rates; tolerance of physical factors; behavior; resource or substrate use; and resistance to disease, parasitism, or predation. Ecological impacts of transgenic fishes are uncertain but will depend primarily on their altered phenotypes and secondarily on the scale and frequency of their introduction into ecosystems. Production of ecologically noxious transgenic types is possible, because many fish varieties readily disperse and persist in aquatic ecosystems, are fit in natural settings, interact substantially with other organisms, and play a role in ecosystem processes. Releases of transgenic fishes may impact aspects of fisheries management. Because future uses of transgenic fish in commercial aquaculture or in stocking of natural waters are contemplated, interdisciplinary teams of fisheries biologists, ecologists, and resource managers must develop integrated approaches to assessing risks of ecological impacts. The current situation of uncertainty demands caution in expecting "safe" uses of transgenic fishes.

Transgenic fish: present status and future directions

Successful production of transgenic fish by gene transfer technology is a very important breakthrough in the techniques of genetic manipulation in animals. This will have an impact of an unprecedented scale in fish biology, aquaculture and mariculture. This is a summary of the workshop on the Transgenic Fish presented at this Symposium. The Workshop discussed the current knowledge, experimental difficulties and related topics of the transgenic fish. It recommended further research on better gene constructs, methods development, safety containment and the closer collaboration of researchers of different disciplines.